CN104530239A - Monoclonal antibody and enzyme linked immunosorbent assay and kit for detecting promethazine drugs - Google Patents

Abstract

The invention discloses a specific monoclonal antibody for detecting various promethazine drugs such as anti-chlorpromazine, anti-promazine, anti-perphenazine drugs and an enzyme linked immunosorbent assay and kit for detecting the promethazine drugs. The monoclonal antibody disclosed by the invention is secreted from a hybridoma 3A5 with the preservation number of CCTCC NO: 201354. Compared with the prior art, the monoclonal antibody disclosed by the invention can be used for identifying various promethazine drugs simultaneously. The enzyme linked immunosorbent assay and kit disclosed by the invention have the advantages of being high in detection efficiency and sensitivity, good in precision and accuracy and the like.

Description

For detecting the monoclonal antibody of phenothiazines and enzyme-linked immunoassay method and test kit

Technical field

The invention belongs to drug testing analysis and immunological technique field, being specifically related to a kind of monoclonal antibody of anti-phenothiazines, the invention still further relates to the enzyme-linked immunoassay method for detecting phenothiazines and test kit.

Background technology

Phenothiazines (Phenothiazines) is the medicine that a class acts on central nervous system, has the mother nucleus structure of sulphur naphthazin(e), can block dopaminergic neuron D ₂acceptor, effects such as there is tranquilizing soporific, reduce body temperature, fatten.Usually use, to reduce pressure production food animal is transported in the process of slaughterhouse.In addition, such medicine Chang Zuowei fodder additives, can improve the feedstuff-meat ratio of animal; Secondly also there is effects such as reducing body temperature, town is told.Therefore, a lot of illegal retailer arbitrarily adds this type of forbidden drug or does not observe the off-drug period, causes medicine residual in edibility animal tissues.Toxicologic study data shows, phenothiazines all can have side effects to nerve system of human body, cardiovascular systems, the recycle system, and eater also can be caused to produce toxic action at a specified future date and potential carcinogenic, teratogenesis, mutagenesis.European Union does not formulate the maximum residue limit (MRL) of this type of medicine, but forbids that this kind of medicine is for food animal.The Ministry of Agriculture of China, the Ministry of Health, National Drug Administration explicitly point out in " the forbidding the types of drugs catalogue used in feed and animal drinking water " of within 2002, combining issue: forbid chlorpromazine to be added in animal-feed and drinking-water, allow therapeutic dose, but must not detect in animal food.

At present, the instrumental method that phenothiazines remains is sensitive, accurate, resolution is high and can carry out the qualitative, quantitative of multi-residue determination, but needs expensive instrument, loaded down with trivial details pre-treatment, skilled professional operation person.Immune analysis method overcomes the defect of instrument analytical method, especially ELISA method is simple to operate, cost is low, highly sensitive, only need simple instrument, be widely adopted at present.

CN101358967 discloses a kind of method of chlorine detection promazine, and vetrnquil and oxammonium hydrochloride are obtained by reacting haptens by it, and prepared monoclonal antibody can only identify chlorpromazine, to the equal nonrecognition of phenothiazines other drug.CN103554056A discloses a kind of phenothiazines haptens, it 2-chloro phenothiazine and bromoacetic acid sodium is reacted to be prepared from, the monoclonal anti physical efficiency obtained identifies chlorpromazine, trilafon, promethazine, Fluphenazine, vetrnquil, thioridazine 6 kinds of phenothiazines simultaneously, and the kind of identification is still insufficient.(the Lowry P. such as Lowry P, Benchikh M.E., Mcconnell R.I., Tohill A., Fitzgerald S.P.Development of a highly sensitive, generic polyclonal antibody for the detection of phenothizines [J] .Life sciences.2010.) utilize vetrnquil and bovine thyroid albumen coupling to obtain immunogen, prepared polyclonal antibody has identification to chlorpromazine, promazine and vetrnquil, but polyclonal antibody variability is large, poor stability.

Summary of the invention

First object of the present invention is to provide a kind of monoclonal antibody that simultaneously can identify multiple phenothiazines;

Second object of the present invention utilizes this monoclonal antibody, prepares a kind of enzyme linked immunological kit for detecting phenothiazines.

3rd object of the present invention utilizes this test kit, sets up a kind of enzyme-linked immunoassay method detected for phenothiazines non-diagnostic object.

Above-mentioned purpose is achieved through the following technical solutions:

A monoclonal antibody for anti-phenothiazines, it is by preserving number secreted by the hybridoma 3A5 of CCTCC NO:C201354.

Described phenothiazines, refers to chlorpromazine, promazine, vetrnquil, thioridazine, Triflupromazine, trilafon, Fluphenazine, chlorprothixene, propiopromazine, promethazine.

Described hybridoma 3A5 is deposited in China typical culture collection center, and preserving number is CCTCC NO:C201354.

Preparing described monoclonal antibody immunogen used is react by trilafon and Succinic anhydried the haptens generated, and prepare with human serum albumin (HSA) coupling, described haptenic chemical structure is as shown in formula I.

Further, the invention provides and a kind ofly detect the residual enzyme-linked immunoassay method of phenothiazines in meat products, the method comprises the following steps:

(1) trilafon and Succinic anhydried are obtained by reacting haptens, described haptenic chemical structure is as shown in formula I;

(2) described haptens and ovalbumin coupling are obtained coating antigen;

(3) monoclonal antibody is prepared with the hybridoma 3A5 that preserving number is CCTCC NO:201354;

(4) use the coating antigen bag of step (2) by solid phase carrier;

(5) enzyme linked immunosorbent detection is carried out after being extracted by testing sample.

Preferably, the extracting method of described testing sample is: by testing sample volume ratio be HCl=4: 1 of acetonitrile: 1M extracting solution extract, centrifugal, supernatant liquor mixes with 0.1M NaOH solution, and then add methylene dichloride, after vortex 5min, recentrifuge, gets dichloromethane layer, dries up and use PBST damping fluid again to dissolve with nitrogen.

The present invention using said monoclonal antibody and coating antigen as core reagent with other conventional agent combination, make the enzyme-linked immunologic detecting kit that can detect phenothiazines, in conjunction with above-mentioned enzyme-linked immunoassay method, achieve the enzyme linked immunosorbent detection to phenothiazines.

The invention has the beneficial effects as follows:

1. the present invention has prepared a kind of monoclonal antibody that simultaneously can identify the multiple phenothiazines such as chlorpromazine, promazine, vetrnquil, thioridazine, Triflupromazine, trilafon, Fluphenazine, chlorprothixene, propiopromazine, promethazine, and the monoclonal antibody prepared by prior art cannot identify above medicine simultaneously.

2. the monoclonal antibody that prepared by the present invention has higher identification sensitivity to above phenothiazines, the IC of chlorpromazine ₅₀be only 0.23 μ g/L, the IC of promazine ₅₀be only 0.22 μ g/L, thioridazine is 0.46 μ g/L, identifies that sensitivity is better than existing phenothiazines monoclonal antibody.And have higher cross reacting rate to above several phenothiazines, and very low to other phenothiazines cross reacting rate, illustrate that there is good specificity.

3. the ELISA method set up of the present invention and test kit can residual in meat products of the multiple phenothiazines such as chlorine detection promazine, promazine, vetrnquil, thioridazine, Triflupromazine, trilafon, Fluphenazine, chlorprothixene, propiopromazine, promethazine simultaneously, method accuracy is high, precision good, once measure and can complete, compared with the existing methods, the kind and efficiency of detection of drugs has obvious advantage.

4. sample extraction method involved in method is simple, fast, simple to operate, the organic reagent used is acetonitrile and methylene dichloride, less to harm.

Accompanying drawing explanation

Fig. 1 is haptens (trilafon-Succinic anhydried reactant), human serum albumin (HSA), immunogenic UV scanning collection of illustrative plates prepared by the present invention.

Fig. 2 be the present invention prepare haptens (trilafon-Succinic anhydried reactant), ovalbumin (OVA), coating antigen UV scanning collection of illustrative plates.

Fig. 3 is the indirect competitive ELISA response curve of monoclonal antibody of the present invention and chlorpromazine (CPZ) standard substance, X-axis is chlorpromazine concentration of standard solution logarithmic value, and Y-axis is that the optical density value of chlorpromazine standard solution is divided by " 0 " hole optical density value (B/B0).

Embodiment

Below by embodiment, the invention will be further described, but do not limit the present invention.

The preparation of embodiment 1 immunogen and coating antigen

The preparation of immunogen/coating antigen

Take trilafon 40mg and Succinic anhydried 20mg, be dissolved in pyridine 10mL, reacting by heating 4h.After adding the distilled water of 20mL precooling, regulate pH to be acid with 2mol/L HCl, add extraction into ethyl acetate, repeatedly carry out 3 times, collect reaction solution evaporate to dryness, namely obtain haptens.

Redissolve with DMF 2mL and walk product, add N, N'-dicyclohexylcarbodiimide (DCC) 30mg and N-hydroxy-succinamide (NHS) 20mg, activation 24h.

Taking HSA 140mg and OVA 200mg is dissolved in 15mL PBS respectively, and under condition of ice bath, be slowly added drop-wise in protein solution by upper step reaction solution, 4 DEG C of reactions are spent the night, and obtain immunogen and coating antigen respectively.Reaction process is as follows:

The preparation of embodiment 2 monoclonal antibody

The preparation of hybridoma: with reference to Du Nianxing " veterinary immunology ".

With the immunogen immune Balb/C mouse (purchased from Hubei Prov. Academy of Medical Sciences's Experimental Animal Center) of preparation in embodiment 1.Immune programme for children is: the neck dorsal sc that containing 100 μ g immunogenic protein emulsion be injected in mouse of fundamental immunity with isopyknic Freund's complete adjuvant emulsification, carries out booster immunization every 15 days with the immunogenic protein emulsion of 100 μ g that contains of Freund's incomplete adjuvant emulsification later.From immunity three times, within the 8th day, adopt tail blood, separation of serum after each immunity, indirect elisa method detects serum antibody titer.The mouse of immuno-competent (height of tiring, sensitivity good) stops immunity in order to merging.

At first three sky of fusion (be better than most immunity terminate rear rest and reorganization January) abdominal injection, reinforced immunological, antigen amount doubles, and does not add adjuvant.

During fusion, mouse orbit sacrificed by exsanguination (collect serum, be positive serum), soaks 5min sterilization in 75% alcohol.Aseptic taking-up mouse spleen, isolates splenocyte.Get 2-5 × 10 ⁷individual myeloma cell mixes with immune spleen cell, and the centrifugal 10min of 1700r/min, abandons supernatant, and back-off is after sterilizing thieving paper control solid carbon dioxide part, and obtained cell mixing, is placed in water-bath.Fusogen (50%PEG) 0.8mL drawing 37 DEG C of incubations slowly adds to cell mixing, and dropping limit, limit is stirred gently, and the fusion time is no more than 1min.The RPMI-1640 basic medium 40mL slowly adding pre-temperature to 37 DEG C stops fusion reaction, and the centrifugal 5min of 800r/min, abandons supernatant.Lower floor's fused cell layer is transferred in the perfect medium containing feeder cell, stirs gently and cell is uniformly distributed.Fused cell suspension is inoculated in 96 porocyte culture plates by 2, every hole, is placed in 37 DEG C of CO ₂cultivate in incubator.

Merge and be designated as 0d the same day, 3d adds in every hole 1 1%HAT complete culture solution, observes colony growth situation.5d tracing observation fused cell, every hole sucking-off 100 μ L supernatant, adds 2 0.5%HAT complete culture solutions, changes liquid once later every 2d.

According to the growing state of cell, when Growth of Cells detects supernatant (usual 7-8d) to when accounting for 1/5-1/3 at the bottom of hole.Get cells and supernatant, screen by indirect competitive ELISA method, 0 hole and medicine hole are set.Compared with 0 hole OD value, medicine hole OD value can be judged to be positive hole in remarkable repressed hole.According to inhibiting rate and cell colony upgrowth situation, select the cell hole only having 1-2 single colony of 2-6 strong positive, adopt limited dilution method to carry out cloning.

Through 3-4 time cloning, until clone's positive rate is 100%, finishing screen selects the hybridoma cell strain of the monoclonal antibody of secreting anti-phenothiazines, and dyed body counting, the chromosome number of this hybridoma is 104.9.Applicant by its called after hybridoma 3A5, and delivers China typical culture collection center (CCTCC) preservation being positioned at Wuhan City, Hubei Province Wuhan University on April 16th, 2013, deposit number is CCTCC NO:201354.

The preparation of ascites monoclonal antibody and qualification: by this cell strain through abdominal injection Balb/C mouse, manufacture order clonal antibody.According to the operational requirement of ThermoScientific company mouse monoclonal antibody Rapid ELISA homotype detection kit, carry out hypotype qualification to the monoclonal antibody that the present invention obtains, result is mouse IgG ₁, light chain is Kappa.

The foundation of embodiment 3 racing ELISA detecting method

The preparation (reagent that the present embodiment uses all adopts following methods preparation except another indicating) of 3.1 reagent

Phosphate buffered saline buffer: NaCl 8.0g, KH ₂pO ₄0.2g, Na ₂hPO ₄.12H ₂o 2.9g, KCl 0.2g, adds distilled water to 1000mL, regulates pH to 7.4;

Coating buffer: get Na ₂cO ₃1.59g, NaHCO ₃2.93g, adds tri-distilled water to 1000mL, adjust ph to 9.6;

Washings: NaCl 8.0g, KH ₂pO ₄0.2g, Na ₂hPO ₄.12H ₂o 2.9g, KCl 0.2g, Tween 200.5mL, add distilled water to 1000mL, regulates pH to 7.4;

Confining liquid: ovalbumin 1g is dissolved in 100mL phosphate buffered saline buffer;

Substrate solution A:3,3', 5', 5-tetramethyl biphenyl diamines (TMB) 200mg, dehydrated alcohol 100mL, add distilled water to 1000mL;

Substrate solution B:Na ₂hPO ₄14.6g, citric acid 9.3g, 0.75% Urea Peroxide 6.4mL, adds distilled water to 1000mL;

Substrate cocktail: by A liquid and B liquid by volume 1:1 mix and get final product, now with the current;

Stop buffer: 2mol/L sulphuric acid soln.

Tentatively determining of 3.2 coating antigen concentration and antibody working concentration

Select the coating antigen of above-mentioned synthesis, be diluted to 8 concentration such as 16mg/L, 8mg/L, 4mg/L, 2mg/L, 1mg/L, 0.5mg/L, 0.25mg/L, 0.125mg/L with coating buffer, at 96 hole enzyme plates, the from first to the 8th leu adds, and 4 DEG C are spent the night; Wash 3 times, pat dry, add confining liquid 250 μ L, 37 DEG C of closed 1h; Wash 3 times, pat dry, the first row to the 8th row of enzyme plate add successively 100 μ L phosphate buffered saline buffers dilution extension rate be 1000,2000,4000,8000,16000,32000,64000,128000 monoclonal antibody, hatch 30min for 37 DEG C, wash 3 times, pat dry; The sheep anti-mouse igg antibody that the horseradish peroxidase (HRP) that each hole adds 1:5000 times of phosphate buffered saline buffer dilution marks (is called for short two to resist, the anti-sheep anti-mouse igg antibody being HRP mark of following indication two, purchased from Wuhan Fei Yi Bioisystech Co., Ltd) 100 μ L, hatch 30min for 37 DEG C, wash 5 times, pat dry; Each hole adds 100 μ L Substrate cocktail, and lucifuge colour developing 15min, adds 50 μ L stop buffers, measure optical density value (OD value), the results are shown in Table 1 by automatic microplate reader at 450nm wavelength place.Result shows, tentatively determine that the bag of coating antigen is 4mg/L or 2mg/L by concentration, antibody working concentration is 1:60000 or 1:30000.

Tentatively determining of table 1 coating antigen concentration and antibody working concentration

The determination of 3.3 best coating antigen concentration

Take chlorpromazine as competitor, be set to 0.6 μ g/L, 0.3 μ g/L, 0.15 μ g/L, 0.075 μ g/L, 0.0375 μ g/L, 0 μ g/L, 6 concentration gradients, the bag selected with 3.2 square formation volumetrys is respectively combined into row indirect competitive ELISA by the antibody dilution of concentration and correspondence.Using the logarithmic value of competitor concentration as X-coordinate, B/B0 (with without OD value during Drug inhibition for B0, OD value during respective concentration Drug inhibition is B value) as ordinate zou draw suppress curve.The results are shown in Table 2, with " 0 " hole OD value and IC ₅₀value, as Judging index, determines best coating antigen concentration.Along with bag is reduced by concentration, IC ₅₀also reduce gradually, therefore from data, best bag is 2 μ g/mL by concentration, and antibody dilution is tentatively defined as 1:30000.

The determination of the best coating antigen concentration of table 2

Coating antigen concentration (μ g/mL)	Antibody dilution multiple (1:X)	0 hole OD value	IC 50Value (μ g/L)
				2	30000	2.121	0.26
4	60000	1.956	0.29

The determination of 3.4 best bag antibody working concentrations

With the best bag by concentration 2 μ g/mL coated elisa plate, antibody concentration equal difference centered by 1:30000 is designed several dilution gradient, its 0 hole and IC ₅₀value is in table 3.Along with the increase of antibody dilution, IC ₅₀value reduces, but considers " 0 " hole value, therefore selects 1:32000 to be optimum antibody extent of dilution.

The determination of table 3 optimum antibody working concentration

Antibody dilution multiple (1:X)	0 hole OD value	IC 50(μg/L)
			28000	2.334	0.34
30000	2.152	0.27
			32000	1.985	0.24
34000	1.726	0.22

The foundation of 3.5 typical curves

With methyl alcohol, chlorpromazine Pharmaceutical formulations is become the mother liquor of 1mg/mL, then with PBS, it is diluted to 6 concentration such as 0,0.0375,0.075,0.15,0.3,0.6 μ g/L successively, carries out indirect competitive ELISA, drawing standard curve, calculate IC ₅₀.As shown in Figure 3, the regression equation of typical curve is y=-0.5744x+0.0987, R ²=0.9953, IC ₅₀value is 0.23 ± 0.01 μ g/L (n=5), and linearity range is 0.0375-0.6 μ g/L.

3.6 cross reaction tests

Suitable concentration gradient is become to carry out indirect competitive ELISA the phenothiazines standard substance doubling dilutions such as chlorpromazine, promazine, thioridazine, Triflupromazine, trilafon, vetrnquil, chlorprothixene, propiopromazine, Fluphenazine, promethazine respectively, each medicine 3 repetition, drawing standard curve, calculates IC ₅₀value, with chlorpromazine standard substance IC ₅₀value contrast obtains cross reacting rate, and result table 4 shows, the indirect competitive ELISA method that the present invention sets up and the cross reacting rate of test kit to these medicines are respectively 100%, 105%, 50%, 23%, 19%, 15%, 11%, 3%, 2%, 2%.

Table 4 test kit of the present invention is to the cross reacting rate of various phenothiazines

Medicine name	IC 50(μg/L)	Cross reacting rate (%)
			Chlorpromazine	0.23	100
Promazine	0.22	105
			Thioridazine	0.46	50
Triflupromazine	1.00	23
			Trilafon	1.18	19
Vetrnquil	1.58	15
			Chlorprothixene	2.20	11
Propiopromazine	9.09	3

Fluphenazine	10.2	2
			Promethazine	11.4	2

This monoclonal antibody all has recognition capability to above 10 kinds of phenothiazines, wherein 7 kinds of medicine IC ₅₀value, at 3 below μ g/L, has very high sensitivity.

Embodiment 4 phenothiazines detects the assembling of ELISA kit

4.1 ELISA kit of the present invention are made up of following part:

(1) solid phase carrier (enzyme plate) of coating antigen is coated with;

(2) 6 bottles, chlorpromazine standardized solution, concentration is respectively 0.6 μ g/L, 0.3 μ g/L, 0.15 μ g/L, 0.075 μ g/L, 0.0375 μ g/L, 0 μ g/L;

(3) preserving number is the monoclonal antibody of the hybridoma 3A5 secretion of CCTCC NO:201354;

(4) the sheep anti-mouse igg antibody working fluid that marks of horseradish peroxidase (HRP);

(5) concentrated phosphoric acid salt buffer: NaCl 80.0g, KH ₂pO ₄2.0g, Na ₂hPO ₄12H ₂o 29.0g, KCl 2.0g, adds distilled water to 1000mL;

(6) concentrated cleaning solution: NaCl 80.0g, KH ₂pO ₄2.0g, Na ₂hPO ₄12H ₂o 29.0g, KCl 2.0g, Tween205mL, add distilled water to 1000mL;

(7) substrate solution A:3,3', 5', 5-tetramethyl biphenyl diamines (TMB) 200mg, dehydrated alcohol 100mL, add distilled water to 1000mL;

(8) substrate solution B:Na ₂hPO ₄14.6g, citric acid 9.3g, 0.75% hydrogen peroxide urea 6.4mL, adds distilled water to 1000mL, regulates pH to 5.0-5.4;

(9) stop buffer: 2mol/L sulphuric acid soln.

The preparation of 4.2 enzyme plates

With coating buffer, coating antigen is diluted to 2mg/L, every hole adds 100 μ L, and 4 DEG C are spent the night, and incline coating buffer, every hole adds 250 μ L washingss and washs 3 times, pat dry, then every hole adds confining liquid 250 μ L, hatches 60min for 37 DEG C, incline liquid in hole, washings washs 3 times, pats dry, and preserves with masking foil vacuum-sealing.

The mensuration program of embodiment 5 enzyme linked immunological kit

The preparation of 5.1 reagent

(1) 0.1M NaOH solution: take 4.2g analytical pure sodium hydroxide, add distilled water and be settled to 1000mL.

(2) sample diluting liquid: use after the concentrated phosphoric acid salt buffer tri-distilled water provided in test kit is diluted 10 times.

(3) washings: use after the washings tri-distilled water provided in test kit is diluted 10 times.

(4) Substrate cocktail: according to each institute expense, by the substrate solution A of preparation and substrate solution B by volume 1:1 mixing, now with the current.

5.2 sample pre-treatments

(1) take pig muscle or the equal pledge 2.0 ± 0.02g of liver specimens in 50mL centrifuge tube, add the extracting solution 4mL that volume ratio is HCl=4: 1 of acetonitrile: 1M, after vortex 5min, the centrifugal 10min of room temperature 4000rpm.

(2) get supernatant liquor 2mL to mix with 4mL 0.1M NaOH, then add methylene dichloride 10mL, after vortex 5min, the centrifugal 10min of room temperature 4000rpm.

(3) get dichloromethane layer 5mL, 50 DEG C of nitrogen dry up, and add after PBST 1mL fully dissolves, for kit measurement.The extension rate of present method to sample is 8.

5.3 determination step

(1) application of sample: add chlorpromazine series concentration standardized solution or sample solution 50 μ L in enzyme plate micropore, then add monoclonal antibody working fluid 50 μ L, be placed in wet box, 37 DEG C of constant-temperature incubation 30min;

(2) wash: pour out the liquid in hole, add washings 250 μ L in every hole and wash 3 times and pat dry;

(3) the sheep anti-mouse igg antibody working fluid that horseradish peroxidase (HRP) marks is added: in every hole, add the sheep anti-mouse igg antibody working fluid 100 μ L that horseradish peroxidase (HRP) marks, be placed in wet box, 37 DEG C of constant-temperature incubation 30min;

(4) wash: pour out the liquid in hole, in every hole, add washings 250 μ L, wash 3 times and pat dry;

(5) substrate is added: add Substrate cocktail 100 μ L in every hole, be placed in wet box, 37 DEG C of constant-temperature incubation 15min;

(6) stop buffer is added: in every hole, add stop buffer 50 μ L;

(7) measure: the optical density value (OD value) measuring every hole by microplate reader at 450nm place.

5.4 results judge

Typical curve:

With measured standard substance OD value divided by " zero " hole OD value (B/B0) for ordinate zou, the logarithmic value of chlorpromazine concentration is that X-coordinate makes typical curve, line linearity of going forward side by side return, provide regression equation.

Phenothiazines concentration calculates:

The inhibiting rate (the OD value of the sample obtained is divided by " zero " hole OD value) of calculation sample, substitute in the regression equation of typical curve, and be multiplied by dilution factor 8, calculate the concentration (μ g/kg) of chlorpromazine in testing sample, be converted to the concentration of phenothiazines other drug according to formula 1.

The sensitivity of embodiment 6 test kit, preci-sion and accuracy

The sensitivity of 6.1 test kits of the present invention

With the IC of typical curve ₅₀value and organize lowest detectable limit (LOD) as the sensitivity index of detection kit of the present invention.The dilution of chlorpromazine standard substance is become 0.6 μ g/L, 0.3 μ g/L, 0.15 μ g/L, 0.075 μ g/L, 0.0375 μ g/L, 0 μ g/L, 6 concentration, the multiple hole of each concentration 5, according to indirect competitive ELISA method replication 5 times, get the IC measured for 5 times ₅₀mean value.LOD is determined by following steps, measures the OD value of 20 parts of pig muscles and liver specimens, goes out corresponding chlorpromazine concentration, then calculate the mean value of chlorpromazine concentration according to the regression equation calculation of typical curve with standard deviation (SD), according to formula (formula 2 calculates the lowest detectable limit in tissue.IC of the present invention ₅₀value is 0.23 ± 0.01 μ g/L, and the lowest detectable limit of phenothiazines in pig muscle and liver refers to table 5.

The lowest detectable limit of phenothiazines in table 5 pig muscle and liver

The precision of 6.2 test kits of the present invention

Chlorpromazine standard substance are diluted to 0.6 μ g/L, 0.3 μ g/L, 0.15 μ g/L, 0.075 μ g/L, 0.0375 μ g/L, 0 μ g/L, 6 concentration, the multiple hole of every concentration 5, according to indirect competitive ELISA method replication 5 times, the regression equation calculation of application standard curve goes out the measured value of each concentration chlorpromazine standardized solution, the variation coefficient in computing board and between plate, the results are shown in Table 6.

Error in the plate of table 6 typical curve and between plate

The accuracy of 6.3 test kits of the present invention, replica test

In 2g homogenate pig muscle or liver organization, add chlorpromazine, promazine, thioridazine standardized solution, make wherein concentration be respectively 0.15 μ g/kg, 0.3 μ g/kg, 0.6 μ g/kg, each concentration 5 repetition, replication 3 times.Measure the concentration of the chlorpromazine of adding in tissue, promazine, thioridazine, calculate the rate of recovery according to the following equation, the accuracy of examination test kit; Calculate within-run and between-run analysis coefficient, the repeatability of examination test kit.The results are shown in Table 7,8, its TIANZHU XINGNAO Capsul, between 76.0-118.1%, with interassay coefficient of variation≤12.3% in batch, shows that this test kit has reliable accuracy, reproducible.

TIANZHU XINGNAO Capsul in table 7 pig muscle

TIANZHU XINGNAO Capsul in table 8 pig liver

Claims (8)

1. the monoclonal antibody of an anti-phenothiazines, it is that described phenothiazines is chlorpromazine, promazine, vetrnquil, thioridazine, Triflupromazine, trilafon, Fluphenazine, chlorprothixene, propiopromazine, promethazine by preserving number secreted by the hybridoma 3A5 of CCTCC NO:C201354.

2. the hybridoma 3A5 described in claim 1, is deposited in China typical culture collection center, and preserving number is CCTCC NO:C201354.

3. monoclonal antibody according to claim 1 detects the application in the enzyme linked immunological kit of phenothiazines in preparation.

4. comprise the test kit of monoclonal antibody according to claim 1.

5. test kit according to claim 4, this test kit is the enzyme linked immunological kit detecting phenothiazines.

6. the application of the test kit described in claim 4 or 5 in phenothiazines non-diagnostic object detects.

7. detect the enzyme-linked immunoassay method that in meat products, phenothiazines is residual, comprise the following steps:

(1) trilafon and Succinic anhydried are obtained by reacting haptens, described haptenic chemical structure is as shown in formula I;

(2) described haptens and ovalbumin coupling are obtained coating antigen;

(3) monoclonal antibody is prepared with the hybridoma 3A5 that preserving number is CCTCC NO:201354;

(4) use the coating antigen bag of step (2) by solid phase carrier;

(5) enzyme linked immunosorbent detection is carried out after being extracted by testing sample,

8. the enzyme-linked immunoassay method that in detection meat products according to claim 7, phenothiazines is residual, it is characterized in that: the extracting method of described testing sample is: by testing sample volume ratio be HCl=4: 1 of acetonitrile: 1M extracting solution extract, centrifugal, supernatant liquor mixes with 0.1M NaOH solution, and then add methylene dichloride, after vortex 5min, recentrifuge, gets dichloromethane layer, dries up and use PBST damping fluid again to dissolve with nitrogen.